A defining feature of presynaptic terminals is the presence of synaptic vesicles (SVs), secretory organelles that store and secrete neurotransmitters. SVs functions are specified by the organelle membrane protein composition. Thus, mechanisms controlling SV formation and composition are pivotal for synapse function. In this proposal we focus on the vesicle biogenesis pathway controlled by the adaptor complex AP-3, a coat complex that sorts membrane proteins from early endosomes to SVs. SV protein composition is regulated by two isoforms of the adaptor complex AP-3, neuronal and ubiquitous, the later thought to participate exclusively in lysosome biogenesis. Genetic defects in the neuronal AP-3 isoform hinder targeting of SV membrane proteins. Surprisingly, null mouse mutants in a lysosomal sorting pathway, the ubiquitous AP-3 route, trigger accruement of SV-specific proteins in SVs. These unexpected results lead us to propose the novel concept that SV and lysosomal sorting mechanisms present on the same endosome compete for membrane proteins to be delivered into two alternative routes, SVs or lysosomes. This concept departs from the traditional view of lysosomes, which are viewed as terminal organelles involved in the disposal of normal and pathological cellular components. Furthermore, our model provides a novel way to understand the contribution of lysosome targeting mechanisms to familial and sporadic forms of neurodegeneration that affect children and adult individuals. Our central hypothesis is that: AP-3-isoform-specific mechanisms target SV membrane proteins from a common endosomal compartment to two competing pathways: either to a SV biogenesis route or to a late endosome-lysosomal path. In this proposal, we will focus on four predictions derived from our hypothesis. These predictions will be systematically explored using a combination of mouse deficient models that affect SV and endo- lysosomal targeting, high-resolution immuno-electron and in vivo imaging microscopy, as well as the molecular analysis of isolated SVs and endosomes. Information gained in this proposal will illuminate our understanding of how late endosomes-lysosome sorting processes affect synapses under physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: We propose a model that will contribute knowledge to understand how lysosomes and synapses interface and contribute to neurodegenerative and psychiatric disorders.